Adeno associated viruses (AAVs) require helper viruses such as adenovirus or herpes virus to achieve productive infection. In the absence of helper virus functions, AAV integrates (site-specifically) into a host cell's genome, but the integrated AAV genome has no pathogenic effect. The integration step allows the AAV genome to remain genetically intact until the host is exposed to the appropriate environmental conditions (e.g., a lytic helper virus), whereupon it re-enters the lytic life-cycle. Samulski (1993) Current Opinion in Genetic and Development 3:74-80 and the references cited therein provides an overview of the AAV life cycle.
AAV-based vectors are used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures. See, West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski (supra) for an overview of AAV vectors.
Recombinant AAV vectors (rAAV vectors) deliver foreign nucleic acids to a wide range of mammalian cells (Hermonat & Muzycka (1984) Proc Natl Acad Sci U.S.A. 81:6466-6470; Tratschin et al. (1985) Mol Cell Biol 5:3251-3260), integrate into the host chromosome (McLaughlin et al. (1988) J Virol 62:1963-1973), and show stable expression of the transgene in cell and animal models (Flotte et al. (1993) Proc Natl Acad Sci U.S.A. 90:10613-10617). Moreover, unlike retroviral vectors, rAAV vectors are able to infect non-dividing cells (Podsakoff et al. (1994) J Virol 68:5656-66; Flotte et al. (1994) Am. J. Respir. Cell Mol. Biol. 11:517-521). Further advantages of rAAV vectors include the lack of an intrinsic strong promoter, thus avoiding possible activation of downstream cellular sequences, and their naked icosohedral capsid structure, which renders them stable and easy to concentrate by common laboratory techniques.
rAAV vectors are used to inhibit, e.g., viral infection, by including anti-viral transcription cassettes in the rAAV vector. For example, Chatterjee et al. (Science(1992), 258:1485-1488, hereinafter Chatterjee et al. 1) describe anti-sense inhibition of HIV-1 infectivity in target cells using an rAAV vector with a constitutive expression cassette expressing anti-TAR RNA. Chatterjee et al. (PCT application PCT/US91/03440 (1991), hereinafter Chatterjee et al. 2) describe rAAV vectors, including rAAV vectors which express antisense TAR sequences. Chatterjee and Wong (Methods, A companion to Methods in Enzymology (1993), 5:51-59) further describe rAAV vectors for the delivery of antisense RNA. Wong Staal et al. (copending U.S. application Ser. No. 08/442,061 filed May 16, 1995) describe composite rAAV vectors which block infection by a wide range of viruses, including HIV-1, HIV-2, HTLV-1 and HTLV-2.
rAAV vectors have several properties which make them preferred gene delivery systems in clinical settings. They have no known mode of pathogenesis and 80% of people in the United States are currently seropositive for AAV (Blacklow et aI. (1971) J Natl Cancer Inst 40:319-327; Blacklow et al. (1971) Am J Epidemiol 94:359-366). Because rAAV vectors have little or no endogenous promoter activity, specific promoters may be used, depending on target cell type. rAAV vectors can be purified and concentrated so that multiplicities of infection exceeding 1.0 can be used in transduction experiments. This allows virtually 100% of the target cells in a culture to be transduced, eliminating the need for selection of transduced cells.
Despite the promising advantages of rAAV vectors as a tool for gene therapy, one problem in their development for clinical use has been the cumbersome and inefficient techniques available for vector production. Packaging cell lines are not available, so present practice utilizes a helper plasmid to co-transfect adenovirus infected cells with the recombinant vector, where the helper plasmid encodes functions and structural proteins which complement AAV functions and structural proteins not encoded by the rAAV vector. Typically, this results in sub-optimal vector production due to the inefficient co-transfection step (See, Muzyczka, supra, and Kotin, supra for an overview of the difficulties in using rAAV vectors). Quite surprisingly, the present invention solves these and other problems associated with the use of AAV-based vectors.